The project aims to develop ratiometric near-infrared (NIR) fluorescent probes for the precise and quantitative analysis of lysosomal and mitochondrial pH changes in live cells, and for effective monitoring of the mitochondrial delivery to the lysosomes during mitophagy caused by nutrient starvation, neurotoxic aminochrome and drug treatment. We will develop ratiometric near-infrared fluorescent probes for sensitive ratiometric detection of intracellular pH changes by electrically conjugating TPE donors to near-infrared hemicyanine acceptors with spirolactam on-off switches based on a TBET (through-bond energy transfer) strategy, or by incorporating hemicyanine or cyanine dyes into rhodol dyes with spirolactam or spiropyran rings on-off switches based on a ?-conjugation modulation strategy. The water solubility and biocompatibility of the probes will be achieved by introducing long oligo(ethylene glycol) residues or mannose residues through oligo(ethylene glycol) tethered spacers to the probes. Morpholine and triphenylphosphonium residues through an oligo(ethylene glycol) spacer will be attached to fluorescent probes for specific targeting of lysosomes and mitochondria in live cells, respectively. Ratiometric near-infrared fluorescent probes based on the TBET strategy will only display TPE donor fluorescence under neutral and basic pH conditions because the hemicyanine acceptors are non-fluorescent with closed spirolactam ring structures in mitochondrial slightly basic condition (pH 8.0). A lysosomal acidic environment will trigger the opening of the acceptor spirolactam ring structures, significantly increase hemicyanine acceptor fluorescence at 740 nm, and considerably reduce TPE donor fluorescence at 640 nm through high efficiency of through-bond energy transfer from the TPE donors to the hemicyanine acceptors. The ratiometric near-infrared fluorescent probes based on ?-conjugation strategy will only show fluorescence of hemicyanine moieties under neutral and basic pH conditions since rhodol moieties will keep closed spirolactam or spiropyran ring structures in mitochondrial slightly basic condition. A lysosomal acidic environment will trigger the opening of the spirolactam or spiropyran rings ring structures of rhodol moieties, significantly enhance ?-conjugation between hemicyanine and rhodol moieties, and result in ratiometric fluorescence responses to pH changes. The probes will possess advantages such as deep tissue penetration, minimum cell damage and interference from biological autofluorescence, large pseudo-Stokes shifts to overcome measurement errors by excitation and scattered lights, self-calibration ratiometric responses with dual-emission capability, excellent water solubility, high stability, high cell permeability, good biocompatibility, excellent intracellular retention, high selectivity, sensitivity, fast and reversible responses to pH changes. We will apply these fluorescent probes for the precise and quantitative analysis of lysosomal and mitochondrial pH changes in HEK293 cells, neuronal cells, HeLa, and breast cancer cells to investigate pH changes and monitor delivery process of the mitochondria to the lysosome during the mitophagy for insightful understanding of physiological and pathological processes.